1. Field of the Invention
This invention relates to biological diagnostic test kits. The invention particularly relates to stabilizers for chromogenic substrates used in enzyme-linked immunosurbent assays.
2. Description of the Background Art
Biological diagnostic test kits, prepackaged assemblages of assay materials, are gaining in popularity, particularly with the advent of the "home" test kit. These kits can be applied to human, veterinary or agricultural testing to detect a wide variety of conditions, commonly by employing antigen specific antibodies, produced in quantity from clones.
Prior to the introduction of test kits, laboratories typically performed these tests using fresh reagents as they were needed. A problem attendant with test kits however, is reagent destabilization as the reagents are left in storage. The mechanism by which destabilization can gradually occur is not well understood. Although gradual environmental oxidation may be a factor, there are likely other contributing factors as yet not understood which may be promoting the destabilization process. The assembled kits may remain on wholesale or retail shelves for extended periods of time, after which they may be stored for even longer periods by the end user. Moreover, storage conditions may often be harmful, for example, under elevated temperatures. These conditions can damage the reagents, rendering test results difficult to interpret, and generally impairing the reliability of the test.
One form of reagent degradation is the discoloration of chromogenic substances to be used in connection with colorimetric enzyme immunoassays. Chromogenic substances particularly those of the hydrogen donor type have a tendency to destabilize when left in storage over long periods of time. This results in a slight color change, typically a yellowig of the chromogenic which occurs gradually over an extended period. Some chromogenic materials, e.g. tetramethylbenzidene; and have been discovered to destabilize less rapidly than others; nonetheless, even this latter chromogen can undergo some discernible yellowing when left in storage for very long periods, particularly under hot environmental conditions. Destabilization of the chromogenic material resulting in yellowing of the chromogen is undesirable, since it can affect the end color change of the chromogen as it reacts to produce a chromophore during conduct of the immunoassay.
Commonly assigned U.S. Pat. No. 4,503,143 discloses an assay which functions as a test kit. The assay is an ELISA test, using tetramethylbenzidine (TMB) as a chromogenic substance. This assay functions quite well, rendering a clearly visible blue color as a positive test result. However, when the kit is stored for very long periods, for example longer than several years and under hot storage conditions, the TMB solution which is normally clear can develop some discernible yellow tinge. The yellow tinge, although slight, poses a potential disadvantage in that it can cause a green tinge to appear in the normally blue chromophore. The green tinge, if pronounced, could possibly be misinterpreted by an inexperienced user.
U.S. Pat. No. 4,067,690 discloses the addition of minute amounts of hydroxylamine to boiler water to retard corrosion due to dissolved oxygen. The hydroxylamine is intended for use in high pressure--high temperature boiler water. The hydroxylamine functions as an oxygen scavenger under these elevated operating conditions to retard corrosion of boiler metal surfaces, generally iron and steel.
U.S. Pat. No. 4,386,224 is directed to use of N,N-diethylhydroxylamine to stabilize color and inhibit color formation in monoalkyl phenols which frequently discolor with age. The hydroxylamine is added in amounts ranging from about 5 parts per million to about 50 parts per million of the alkyl phenol. This reference also discloses that the N,N-diethylhydroxylamine can reduce the color of monoalkyl phenols in which color development has taken place.
Accordingly, it is an object of the invention to retard degradation and discoloration of a chromogen during storage.
It is an object of the invention to provide a stabilizing agent for the chromogenic solutions to be used in enzyme immunoassays wherein the stabilizing agent retards discoloration of the chromogenic solution over an extended period of time in excess of one year.
It is a further object to provide a stabilizing agent to retard discoloration of chromogenic solutions of the hydrogen donor type to be used in colorimetric enzyme immunoassay kits.